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Abstract

The Zika virus is the primary suspect in the large increase in microcephaly cases in 2015-6 in Brazil, however its role is unconfirmed despite individual cases of viral infections found in neural tissue. Here we consider the alternative that the insecticide pyriproxyfen, used in Brazilian drinking water for mosquito control, may actually be the cause. Pyriproxifen is an analog of juvenile hormone, which corresponds in mammals to regulatory molecules including retinoic acid, a vitamin A metabolite, with which it has cross-reactivity and whose application during development causes microcephaly. Methoprene, another juvenile hormone analog approved as an insecticide has metabolites that bind to the retinoid X receptor, and causes developmental disorders in mammals. Isotretinoin is another example of a retinoid causing microcephaly in human babies via activation of the retinoid X receptor. Moreover, tests of pyriproxyfen by the manufacturer, Sumitomo, widely quoted as giving no evidence for developmental toxicity, actually found some evidence for such an effect, including low brain mass and arhinencephaly--incomplete formation of the anterior cerebral hemispheres--in rat pups. Finally, the pyriproxyfen use in Brazil is unprecedented--it has never before been applied to a water supply on such a scale. Claims that it is not being used in Recife, the epicenter of microcephaly cases, do not distinguish the metropolitan area of Recife, where it is widely used, and the municipality, where it is not. Given this combination of information we strongly recommend that the use of pyriproxyfen in Brazil be suspended pending further investigation.

Author Comment

The cause of over 6,000 suspected microcephaly cases in Brazil is being investigated. The primary suspect is the Zika epidemic. An alternative that has been widely dismissed is the larvicide pyriproxyfen, used in drinking water for mosquito control. Pyriproxifen is a juvenile hormone analog, which has cross-reactivity with retinoic acid, whose application during mammalian development causes microcephaly. Several other juvenile hormone analogs have been shown to cause microcephaly and other birth defects. Despite regulatory approval, toxicology studies are very limited; in the most relevant study one out of twelve exposed rat pups was found to have significantly low brain weight. We advise stopping the use of pyriproxyfen in Brazil pending better determination of its developmental toxicity.

Funding

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An important consideration would be as to the type of binding that pyriproxyfen has to target molecules - e.g. retinoid receptor molecules - if there is high affinity binding low concentrations may still lead to very adverse effects. Also, pyriproxyfen may accumulate in the body - it is fat soluble and evidence in the literature suggests it may also bind to DNA - so may reach a saturation point in sequestration and binding sites if there is continued intake via drinking water where sudden release of significant concentrations into circulation could occur. This occurring during early pregnancy could be very damaging if pyriproxyfen does indeed bind to retinoid receptors.

Kathryn Kitson's comments are very helpful. There is definitely a need for molecular studies of pyriproxyfen's effects in mammals, and humans more specifically. Retinoid receptor binding or activation is only one possible mode of action.

It is clear that there is significant overlap between arthropod and vertebrate hormone signalling mechanisms, and claims for pyriproxyfen's safety made on the basis that juvenile hormone analogs have no effect in vertebrates are not supported by fact.

Another mechanism worth considering as a possible cause of microcephaly is an interaction of pyriproxyfen with thyroid hormone, via synergistic effects of thyroid hormone receptor and retinoid receptor co-activation. It is interesting that thyroid hormone receptor forms a heterodimer with retinoid X receptor and that exposure to retinoids and thyroid hormone are both implicated in the derangement of cranial development and microcephaly. It is possible that pyriproxyfen plays a role in increasing the effects of thyroid hormone via retinoid receptor activation and that maternal TH levels might then also be a contributing factor. This could explain differences in observed effects from similar doses of pyriproxyfen

Yes!! A further possible complication is that pyriproxyfen may also have an affinity for the binding site in the aldehyde dehydrogenase that functions to convert retinal to retinoic acid - if its interaction with the RXR (and perhaps RAR?) receptor is based around a general structural similarity to retinal/retinoic acid - therefore pyriproxyfen may also inhibit the formation of retinoic acid needed to bind retinoid receptors to initiate crucial stages in development - notably facial and head shape, limb formation, and probably also neuronal connections within the developing brain.

Agree, activation of RAR should be considered as a possibility, as should interference with conversion of retinal. Reports of ocular changes and "gross pigment mottling" in the eyes of some of the microcephalic babies in Brazil points to a possible interaction between the causal agent and RAR/RXR

As well as pyriproxyfen, the action of its environmental and biological metabolites also need to be considered. For, example environmental metabolites of the juvenoid methoprene have been shown to have higher toxicity and teratogenic potential than the parent compound. PYPAC is a major breakdown product of pyriproxyfen in water which has not been subjected to any toxicological assessments

0

The authors should be commended for a thorough and nuanced analysis of a pressing health emergency.

However, I am struck by the conclusion that doses in animal models of 100 mg/kg or 300 mg/kg dosage have any relevance to what is almost certainly ppm exposure in humans. Eg, 300 mg/kg for an adult woman (assuming 60 kg) would be 18g of pyriproxyfen!

The issue of exposure levels is indeed relevant. However direct extrapolation of mg/kg are not always appropriate given that in early development the size and weight of human embryos and those of other mammals used in experimental models are similar. Currently accepted toxicological methodologies are not well suited to examine low dose effects.

The published guidelines for application for the larvicide in Brazil suggest that exposure levels are likely to exceed 1mg in some instances.

It is also unclear how the effects of breakdown products of pyriproxyfen in standing water might differ from direct dosing with the parent compound in toxicological studies.

It would be appropriate for pyriproxyfen to be tested in larger cohorts of experimental animals at doses comparable to those likely to be experienced in areas where pyriproxyfen is used as a larvicide in human drinking water supplies. This is a gap in existing safety data. It should also be noted that species specific hormone signalling and embryonic development means that the effects of a hormone mimic such as pyriproxyfen could differ between mammalian species. For example, retinol like effects such as premature cranial suturing may be more apparent in an animal with a larger head and longer gestation times.

Andrew Lover raises important considerations, but the fact that pyriproxyfen has not been tested on humans and that the current mass administration in Brazil is entirely unprecedented means that a more cautious approach should be applied. There is no conclusive evidence in this paper that pyriproxyfen causes microcephaly in humans, but on the basis of currently available data that possibility cannot be precluded. Given that safe alternatives are available, it would be prudent to suspend use of pyriproxyfen in drinking water, pending further investigation

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